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Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Published on: December 20, 2010
Structural and Spectroscopic Basis for Catalysis by a Class C Radical S-Adenosylmethionine Methylase Involved in
Bo Wang1,2,3, Hayley L Knox1, Nicholas J York1
1Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
Abstract:
Nosiheptide (NOS) is a ribosomally synthesized and post-translationally modified peptide natural product that exhibits potent antibiotic activity against multiple bacterial pathogens. NOS features a core macrocyclic peptide containing thiazoles, dehydrated serine and threonine residues, and a 3-hydroxypyridine ring. In addition to the macrocycle, NOS possesses a side-ring system formed by a 3-methyl-2-indolic acid (MIA) bridge that connects to glutamyl and cysteinyl residues on the core peptide via ester and thioester linkages. This unique side-ring is installed by the class C radical S-adenosylmethionine (SAM) methylase NosN. Here, we report three X-ray crystal structures of the NosN homologue, NocN, at resolutions of 1.40 Å, 1.84 Å, and 1.78 Å under anaerobic conditions, representing the first structural characterization of a class C radical SAM methylase. The structures reveal clear electron density for two bound SAM molecules. Remarkably, the C5' atom of SAMI, which coordinates to the [Fe4S4] cluster, lies 3.5 Å from the methyl group of SAMII and is properly positioned for direct hydrogen atom abstraction. A structure containing a product mimic illustrates how NocN engages its substrate and identifies Tyr276 as a key catalytic residue. The structure further suggests that the sulfonium center of SAMII may undergo epimerization to facilitate radical attack. Finally, electron paramagnetic resonance spectroscopy identifies a paramagnetic species consistent with the addition of the SAMII-derived methylene radical to the MIA substrate.
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